Devices including muscle matrix and methods of production and use

ABSTRACT

Tissue compositions and methods of preparation thereof are provided. The tissue compositions can be used to treat or regenerate muscle tissue. The compositions can be configured to provide increased strength compared to other muscle matrices.

This application is a continuation of U.S. patent appliation Ser. No.15/881,911, filed Jan. 29, 2018, which claims priority under 35 USC §119 to U.S. Provisional Application No. 62/451,981, which was filed onJan. 30, 2017. Each of the above applications is incorporated herein byreference in its entirety.

The present disclosure relates to tissue products, and moreparticularly, to tissue matrices produced from muscle tissue.

Various injuries, diseases, and surgical procedures result in the lossof muscle mass, particularly skeletal muscle. For example, surgicalremoval of soft tissue sarcomas and osteosarcomas can result in the lossof bulk muscle. Other surgical and cosmetic procedures, such as herniarepair and muscle augmentation, require long-term management of musclecontent. Muscle damage can also result from injury, such as from bluntforce trauma and gunshot injuries.

Current muscle regenerative procedures focus on the use of muscleallografts (e.g., harvesting gluteus maximus muscle from donor sites onthe patient or from a cadaver) and the use of xenografts comprisingcompletely decellularized dermal or other tissue matrices. However, theuse of muscle transplants can lead to excess inflammation (resulting inscar tissue formation and potential rejection) and, if harvested from apatient, presents the problem of muscle loss at the donor site.

Currently, partially decellularized matrices can be produced foreffective muscle treatment. However, these matrices may not supportsufficient loads and may break down under stress. Thus, a need remainsfor improved methods and compositions for muscle treatment orregeneration.

Taught herein is a method of preparing a tissue composition. The methodincludes providing a tissue sample wherein the tissue sample comprises amuscle portion and a fascia portion harvested without separating themuscle portion of the tissue from the fascia portion of the tissue. Themethod also includes processing the tissue sample to produce at leastone decellularized musculofascial matrix.

Taught herein is a tissue composition comprising at least onedecellularized muscle matrix and at least one decellularized fasciamatrix. The muscle matrix and the fascia matrix comprise at least onemuscle tissue and at least one connected fascia tissue harvested withoutseparating the muscle tissue from the fascia tissue. The muscle matrixcontains at least some of the myofibers normally found in an unprocessedmuscle sample.

Taught herein is a method of preparing a tissue composition includingproviding a muscle sample and processing the muscle sample to produce adecellularized muscle matrix wherein myofibers of the muscle matrix areoriented in a longitudinal direction.

Taught herein is a tissue composition comprising at least onedecellularized muscle matrix that contains at least some of themyofibers normally found in an unprocessed muscle sample and wherein themyofibers are oriented longitudinally.

Taught herein is a method of preparing a tissue composition includingproviding a group of muscle matrices and layering and joining the groupof muscle matrices to produce a multi-layer muscle matrix.

Taught herein is a tissue composition comprising multiple layers ofdecellularized muscle matrix that contain at least some of the myofibersnormally found in an unprocessed muscle sample.

Taught herein is a method of preparing a tissue composition includingselecting a muscle matrix layer and selecting a supporting layer. Themethod also includes applying a slurry comprising particulate acellulartissue matrix (ATM) to at least one of the muscle matrix layers or thesupporting layer. The method also includes joining the muscle matrixlayer and the supporting layer using the slurry.

Taught herein is a tissue composition including at least one musclematrix layer and at least one supporting layer. The tissue compositionalso includes at least one particulate acellular tissue matrix (ATM)that attaches the at least one muscle layer to the at least onesupporting layer.

Taught herein is a method of preparing a tissue composition. The methodincludes selecting a muscle matrix layer and selecting a supportinglayer including one or more pores. The method includes applying a slurrycomprising particulate acellular tissue matrix (ATM) to at least one ofthe muscle matrix layers or the supporting layer. The method includesapplying the supporting layer to a first surface of the muscle matrixlayer. The method also includes rolling the muscle matrix layer and thesupporting layer such that at least a portion of the first surface ofthe muscle matrix layer attaches to at least a portion of a secondsurface of the muscle matrix layer through the one or more pores.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a perspective view of a muscle and fascia tissuesource that may be harvested to create a tissue product.

FIG. 2 illustrates a side view of a layered muscle tissue composition,according to various embodiments.

FIG. 3A illustrates a side view of a tissue composition comprising amuscle layer and a slurry according to various embodiments.

FIG. 3B illustrates a side view of a tissue composition comprising amuscle layer, a supporting layer, and a slurry according to variousembodiments.

FIG. 4 illustrates a side view of a rolled tissue composition comprisinga muscle layer, a supporting layer, and a slurry according to variousembodiments.

FIG. 5 is a bar graph of normalized max load values of porcine loinmuscle cut in different directions and with different orientations ofmyofibers, according to various embodiments of the disclosed inventions.

FIG. 6 is a bar graph of normalized max load values of porcine rectusabdominis muscle cut longitudinally or cross-sectionally according tovarious embodiments of the present disclosure.

FIG. 7 is a bar graph of normalized max load values of porcine loinmuscle with cross sectionally-oriented myofibers and longitudinallyoriented myofibers with and without fascia according to variousembodiments of the present disclosure.

FIG. 8 is a bar graph of normalized max load values of porcine externaloblique muscles cut longitudinally or cross-sectionally and with orwithout fascia according to various embodiments of the presentdisclosure.

FIG. 9 is a bar graph of normalized max load values for porcine musclematrix compositions derived from cross-sectionally cut loin musclepieces that included a single muscle matrix layer or two muscle matrixlayers according to various embodiments of the present disclosure.

FIG. 10 is a bar graph of normalized max load values of single andbilayer porcine muscle matrix compositions derived from longitudinallycut loin muscle pieces measured in the X direction and the Y directionaccording to various embodiments of the present disclosure.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

Various human and animal tissues can be used to produce products fortreating patients. For example, various tissue products forregeneration, repair, augmentation, reinforcement, and/or treatment ofhuman tissues that have been damaged or lost due to diseases and/orstructural damage (e.g., from trauma, surgery, atrophy, and/or long-termwear and degeneration) have been produced. Such products can include,for example, acellular tissue matrices, tissue allografts or xenografts,and/or reconstituted tissues (i.e., at least partially decellularizedtissues that have been seeded with cells to produce viable materials).

As used herein, “myofibers” are the rod-like structures involved inmuscle contraction and comprise proteins such as myosin, troponin,tropomyosin, and actinin. Long myofiber chains are found in and betweenthe elongated muscle cells (myocytes).

As used herein, a “muscle defect” is any muscle abnormality or damagethat is amenable to repair, improvement, enhancement, regeneration,amelioration, and/or treatment by an implanted muscle matrix. A muscledefect encompasses any abnormality or damage resulting from disease,trauma, or surgical intervention that results in an alteration to themuscle. As used herein, the removal or loss of “bulk” muscle tissuerefers to the loss of an appreciable and measurable volume of muscletissue, e.g., a volume of at least about 0.5 cm³.

As used herein, a “decellularized tissue” is any tissue from which mostor all of the cells that are normally found growing in the extracellularmatrix of the tissue have been removed (e.g., a tissue lacking about 80,85, 90, 95, 99, 99.5, or 100% of the native cells, or any percentage inbetween).

The materials and methods provided herein can be used to make abiocompatible implant. As used herein, a “biocompatible” implant is acomposition that has the ability to support the migration andproliferation of native cells from surrounding tissue into thecomposition following implantation and that does not elicit asubstantial immune response that prevents such cellular activity. Asused herein, a “substantial immune response” is one that preventspartial or complete resorption of the implanted material and/or thepartial or complete repopulation of the implant with native cells.

As used herein, the terms “native cells” and “native tissue” mean thecells and tissue present in the recipient tissue or organ prior to theimplantation of a muscle implant, or the cells or tissue produced by thehost animal after implantation.

FIG. 1 is a perspective view of a muscle and fascia tissue source thatmay be harvested to create a tissue sample. In an exemplary tissuesource, muscle tissue 110 is surrounded by fascia tissue 120. The tissuesource may be any human or animal skeletal muscle and accompanyingfascia that is suitable for decellularization and subsequentimplantation in a treatment site. Once implanted, the decellularizedmusculofascial matrix produced from the tissue source can provideincreased strength to the implanted region and/or can promote in-growthof muscle cells and regeneration of muscle tissue.

In exemplary embodiments, tissue compositions and decellularizedmusculofascial matrices described herein can have a higher initialstrength than those known previously and can withstand higher loadswithout tearing. In some embodiments, the fascia matrix can provide ascaffold into which native cells (e.g., fibroblasts, etc.) can migrate,allowing for the remodeling of fascia and/or dermis along with theremodeled muscle induced by the muscle matrix.

In certain embodiments, the tissue sample comprises a muscle portion anda fascia portion harvested without separating the muscle portion 110 ofthe tissue from the fascia portion 120 of the tissue (including some orall of the surrounding fascia). Harvesting muscle tissue withsurrounding fascia tissue enables the preparation of a tissuecomposition that can withstand a higher max load than a tissuecomposition composed of solely or predominately muscle tissue.Preparation of a muscle matrix is described in more detail below and isalso described in pending U.S. application Ser. No. 14/410,204, filed onDec. 22, 2014 and published as US Patent Publication US2015/0282925,which is incorporated herein by reference in its entirety.

In various embodiments, the tissue sample can be processed to removeblood or blood components such as red blood cells. For example, thetissue sample can be exposed to a cell lysis solution to remove cellssuch as red blood cells. A variety of blood cell removal or lysissolutions can be used, including, for example, solutions such asammonium chloride, hypo- or hypertonic-saline, detergents, or other knowblood removal compositions. Further, the solutions can be used in anumber of incubation and/or wash steps, including for example, one toten wash steps, or any suitable number in between.

In various embodiments, the tissue sample can be processed to produce adecellularized musculofascial matrix. For example, the tissue sample canbe exposed to a decellularization solution in order to remove viable andnon-viable cells from the muscle tissue without damaging the biologicaland/or structural integrity of an extracellular matrix within the muscletissue. The decellularization solution may contain an appropriatebuffer, salt, an antibiotic, one or more detergents (e.g., TRITON X100™or other nonionic octylphenol ethoxylate surfactants, sodium dodecylsulfate (SDS), sodium deoxycholate, or polyoxyethylene (20) sorbitanmonolaurate), one or more agents to prevent cross-linking, one or moreprotease inhibitors, and/or one or more enzymes. In some embodiments,the decellularization solution can comprise 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or anyintermediate percentage of TRITON X-100™ and, optionally, 10 mM, 15 mM,20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or any intermediateconcentration of EDTA (ethylenediaminetetraacetic acid). In certainembodiments, the decellularization solution can comprise 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%,or any intermediate percentage of sodium deoxycholate and, optionally, 1mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM,13 mM, 14 mM, 15 mM, or 20 mM HEPES buffer(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing 10 mM,15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or anyintermediate concentrations of EDTA. In some embodiments, the muscletissue can be incubated in the decellularization solution at 20, 21, 22,23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42degrees Celsius (or any temperature in between), and optionally, gentleshaking can be applied at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, or 150 rpm (or any rpm in between). The incubation can befor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36, 48, 60, 72,84, or 96 hours (or any time period in between).

The length of time of exposure to the decellularization solution and/orthe concentration of detergent or other decellularizing agents can beadjusted in order to control the extent of decellularization andmyofiber removal from the muscle tissue. In certain embodiments,additional detergents may be used to remove cells from the muscletissue. For example, in some embodiments, sodium deoxycholate, SDS,and/or TRITON X-100™ can be used to decellularize and separate undesiredtissue components from the extracellular tissue matrix.

In some embodiments, the tissue sample can be contacted with a solutionincluding trypsin in order to break down muscle fiber bundles (e.g., bycleaving myosin molecules in the muscle fiber). In some embodiments, thesolution can include additional enzymes such as papain, bromelain,ficin, or alcalase. In some embodiments, trypsin can facilitate thedecellularization process by increasing the rate and/or extent ofmyofiber breakdown and myocyte removal during subsequentdecellularization. In some embodiments, the muscle sample can be exposedto trypsin at a concentration in a range from about 10⁻¹⁰-0.5% (e.g., atabout 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2,0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 percent), or from 10⁻⁸-10⁻⁴%, or from10⁻⁷-10⁻⁵%, or any percent inbetween. The aforementioned concentrationscan be considered appropriate for enzymes that have an enzymaticactivity such that 10⁻⁶% corresponds to approximately 120-130 BAEEunits, and a BAEE unit is determined for enzymes with a specificationfor trypsin activity using Na-Benzoyl-L-arginine ethyl ester (BAEE) as asubstrate. The procedure is a continuous spectrophotometric ratedetermination (ΔA253, Light path=1 cm) based on the following reaction:

where:

BAEE=Nα-Benzoyl-L-arginine ethyl ester; and

A BAEE Unit is defined such that one BAEE unit of trypsin activity willproduce a ΔA253 of 0.001 per minute with BAEE as substrate at pH 7.6 at25° C. in a reaction volume of 3.20 ml.

A number of suitable trypsins may be used, but one exemplary trypsinthat may be appropriate include bovine pancreatic trypsin, e.g., fromSigma Aldrich (Sigma-Aldrich product T1426).

In certain embodiments, the muscle sample can be exposed to trypsin forat least about 15 minutes or up to a maximum of about 24 hours (e.g.,about 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90minutes, 105 minutes, 120 minutes, 4 hours, 8 hours, 12 hours, 24 hoursor any intermediate time). In certain embodiments, muscle samplesincluding fascia can be exposed to trypsin for at least about 15 minutesor up to a maximum of about 48 hours (e.g., about 15 minutes, 30minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes,120 minutes, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours or anyintermediate time). In various embodiments, decellularization can bedone before trypsinization, after trypsinization, or both before andafter trypsinization.

The procedure to decellularize the tissue sample can, in someembodiments, be controlled to retain at least some myofibers normallyfound in the tissue sample prior to processing. For example, the lengthof exposure and/or the concentration of the decellularization solutionand/or trypsin solution can be adjusted in order to control the extentof myofiber removal. In some embodiments, the duration and/orconcentration are selected in order to remove about 20-80% of themyofibers normally found in the muscle tissue prior to trypsinizationand decellularization. In certain embodiments, the duration and/orconcentration are selected in order to remove about 20, 30, 40, 50, 60,70, 80, or 90 percent of the myofibers (or any percentage in between).In some embodiments, about 20-80% of the myofibers are removed byexposing the tissue sample to trypsin at a concentration ranging from10⁻¹⁰-0.5% for 15 minutes to 24 or 48 hours and/or by exposing themuscle tissue sample to about 0.1-2.0% of a decellularization agent(e.g., TRITON X-100™ or other nonionic octylphenol ethoxylatesurfactant, sodium dodecyl sulfate, sodium deoxycholate, orpolyoxyethylene (20) sorbitan monolaurate) for 0.1-72 hours.

In other embodiments, the procedure to decellularize the tissue samplewhile retaining at least some myofibers normally found in the tissuesample prior to processing can be controlled by adjusting the ratio oftissue mass to volume of decellularization or trypsinization solution(e.g., the mass of tissue per volume of solution containing trypsinand/or decellularizing agents). In some embodiments, a lower ratiotissue to volume of solution can increase the efficiency of the myofiberremoval process, thus resulting in a decellularized musculofascialmatrix that retains fewer intact myofibers. In other embodiments, ahigher ratio of tissue to volume of solution ratio can reduce theefficiency of the myofiber removal process, thus resulting in adecellularized musculofascial matrix that retains more intact myofibers.

In various embodiments, the extracellular scaffold within adecellularized muscle or musculofascial tissue may include collagen(particularly collagen type I or type III), elastin, myofiber, and/orother fibers, as well as proteoglycans, polysaccharides, and/or growthfactors (e.g. IGF, EGF, Ang 2, HGF, FGF, and/or VEGF). The muscle ormusculofascial matrix may retain some or all of the extracellular matrixcomponents that are found naturally in a muscle prior todecellularization, or various undesirable components may be removed bychemical, enzymatic, and/or genetic means. In general, the muscleextracellular matrix provides a structural scaffold comprising fibers,proteoglycans, polysaccharides, and growth factors into which nativecells and vasculature can migrate, grow, and proliferate afterimplantation in a patient. The exact structural components of theextracellular matrix will depend on the type of muscle and/or fasciaselected and the processes used to prepare the decellularized tissue.

In certain embodiments, the tissue sample including muscle and fasciatissue can be chemically treated to stabilize the tissue so as to avoidbiochemical and/or structural degradation before, during, or after cellremoval. In various embodiments, the stabilizing solution can arrest andprevent osmotic, hypoxic, autolytic, and/or proteolytic degradation;protect against microbial contamination; and/or reduce mechanical damagethat can occur during decellularization. The stabilizing solution cancontain an appropriate buffer, one or more antioxidants, one or moreoncotic agents, one or more antibiotics, one or more proteaseinhibitors, and/or one or more smooth muscle relaxants. In someembodiments, the stabilizing solution can include one or more freeradical scavengers including, but not limited to, glutathione,n-acetylcysteine, superoxide dismutase, catalase, or glutathioneperoxidase.

In certain embodiments, a muscle or musculofascial implant can compriseone or more additional agents. In some embodiments, the additionalagent(s) can comprise an anti-inflammatory agent, an analgesic, or anyother desired therapeutic or beneficial agent. In certain embodiments,the additional agent(s) can comprise at least one added growth orsignaling factor (e.g., a small cell growth factor, an angiogenicfactor, a differentiation factor, a cytokine, a hormone, and/or achemokine). These additional agents can promote native muscle migration,proliferation, and/or vascularization. In some embodiments, the growthor signaling factor is encoded by a nucleic acid sequence containedwithin an expression vector. As used herein, the term “expressionvector” refers to any nucleic acid construct that is capable of beingtaken up by a cell, contains a nucleic acid sequence encoding a desiredprotein, and contains the other necessary nucleic acid sequences (e.g.,promoters, enhancers, initiation and termination codons, etc.) to ensureat least minimal expression of the desired protein by the cell.

In certain embodiments, the muscle and/or fascia tissue used to preparea muscle or musculofascial matrix can be treated with one or moreenzymes to remove undesirable antigens, e.g., an antigen not normallyexpressed by the recipient animal and thus likely to lead to an immuneresponse and/or rejection. For example, in certain embodiments, muscleand/or fascia tissue can be treated with alpha-galactosidase to removealpha-galactose (α-gal) moieties. In some embodiments, to enzymaticallyremove a-gal epitopes, after washing the muscle tissue thoroughly withsaline, the tissue may be subjected to one or more enzymatic treatmentsto remove a-gal antigens, if present in the sample. In certainembodiments, the muscle and/or fascia tissue may be treated with ana-galactosidase enzyme to substantially eliminate a-gal epitopes. Inaddition, certain exemplary methods of processing tissues to reduce orremove alpha-1,3-galactose moieties are described in Xu et al., “APorcine-Derived Acellular Dermal Scaffold That Supports Soft TissueRegeneration: Removal of Terminal Galactose-α-(1,3)-Galactose andRetention of Matrix Structure” Tissue Engineering Part A, Vol. 15(7),1807-1819 (2009), which is hereby incorporated by reference in itsentirety.

In some embodiments, the tissue source is porcine. In alternativeembodiments, the tissue source is human. In certain embodiments, themuscle tissue is harvested from skeletal muscle. A decellularizedmusculofascial matrix can comprise muscle tissue from one or more (e.g.,1, 2, 3, 4, 5, or more) different muscles.

While the decellularized muscle tissue in a musculofascial matrix may bederived from one or more donor animals of the same species as theintended recipient animal, this is not necessarily the case. Thus, forexample, the decellularized muscle tissue may be prepared from porcinetissue and implanted in a human patient. Species that can serve asdonors and/or recipients of decellularized muscle tissue include,without limitation, mammals, such as humans, nonhuman primates (e.g.,monkeys, baboons, or chimpanzees), pigs, cows, horses, goats, sheep,dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice. Insome embodiments, muscle tissue from more than one donor animal can beused.

In certain embodiments, animals that have been genetically modified tolack one or more antigenic epitopes may be selected as the tissue sourcefor a muscle matrix. For example, animals (e.g., pigs) that have beengenetically engineered to lack expression of the terminal α-galactosemoiety can be selected as the tissue source. For descriptions ofappropriate animals and methods of producing transgenic animals forxenotransplantation, see U.S. patent application Ser. No. 10/896,594 andU.S. Pat. No. 6,166,288, both of which are hereby incorporated byreference in their entirety.

Muscle or musculofascial implants that include decellularizedmusculofascial matrices as described herein can be produced. Suchimplants can be used to treat various muscle defects and relateddisorders where repair, alteration, regeneration, and/or enhancement ofmuscle tissue is desired. For example, the implants can be used to treathernias and other abdominal wall muscle injuries, where the currentstandard of care generally involves the use of fully decellularizeddermal matrices or intact muscle transplants that are more effective inpromoting fascia regeneration than regeneration of functional muscle. Inanother example, the implants can be used to repair a traumaticabdominal wall injury, such as from a gunshot or other blunt forceinjury. In yet another example, the implants can be used following thesurgical removal of bulk tissue (e.g., after removal of a soft tissuesarcoma or osteosarcoma). In various embodiments, implants can be usedto repair a defect in any type of skeletal muscle including, but notlimited to, gluteus maximus muscle, rectus muscle, bicep femoris muscle,or gastrocnemius muscle.

In some embodiments, an implant can also be used after surgical removalof bulk muscle tissue (e.g., after surgical intervention to remove asarcoma or osteosarcoma). For patients that do not receive an implant orthat receive an implant comprising intact muscle or decellularizedtissue that lacks any remaining myofibers, the rate and overall volumeof muscle repair can be low. Conversely, implants according to thepresent disclosure can initiate and/or improve the rate and overallvolume of muscle repair by inducing a sufficient (but not excessive)level of inflammation that serves to recruit the patient's muscle repairpathways (e.g., macrophage/myoblast recruitment and satellite cellactivation). Similarly, in surgical procedures where muscle and/orfascia tissue is harvested from one muscle and/or fascia fortransplantation into another location on the patient, implants asdescribed herein can be placed at the harvest site to help promote therate and overall extent of muscle and/or fascia repair at the harvestsite following the transplant procedure.

In some embodiments, an implant can be used to enhance native musclevolume. For example, the implant can be used as part of a treatment fora muscle wasting disease, thereby enhancing the rate of repair andregeneration, and/or increasing the overall volume of muscle at theimplant site. In another example, the implant can be used tocosmetically enhance the appearance of muscle tissue by promoting thegrowth of additional muscle volume at the implant site.

When an implant comprising one or more decellularized muscle matricesand/or one or more decellularized fascia matrices is used, the musclematrix in the implant can promote muscle regeneration while the fasciamatrix in the implant can promote repair or regeneration of the nearbyfascia. In contrast, current surgical procedures (e.g., the use ofsutures and/or implanted decellularized tissue matrices that lackmyofibers) can result in repair to the fascia but minimal repair orregeneration of underlying muscle. The resulting lack of underlyingmuscle regeneration in current surgical procedures can lead to anincreased rate of bulging, scarring, and other complications.

Several techniques in addition to or alternative to producing musclematrix with accompanying fascia are disclosed herein to improve thestrength of decellularized musculoskeletal matrices and tissuecompositions. In some embodiments, the orientation of the myofibers inthe tissue sample can affect the strength of the resulting tissuematrix.

In some embodiments, the tissue composition can be prepared by selectingthe tissue sample such that myofibers of the decellularizedmusculofascial matrix are oriented in a particular direction. In someembodiments, the orientation of the cut can be the same as or differentthan the orientation of myofibers within the tissue.

For example, some muscles, such as the rectus muscles, tend to havemyofibers oriented along the long axis or direction of force generationof the muscle; while other muscles, such as the obliques of theabdominal wall or the loin muscles can have fibers oriented obliquely ordifferently with respect to the long axis or direction of forcegeneration of the muscle. In accordance with various embodiments, theorientation of the myofibers in a tissue sample can have an effect onthe maximum load that a resulting tissue composition can withstand.Accordingly, in some embodiments, the present disclosure includesdevices that incorporate tissue matrices formed from muscles cut intosections along selected directions. For example, the devices can includetissue matrices from muscles that are cut longitudinally orcross-sectionally, or for multi-layered devices, can includecombinations of tissue matrix types.

The muscle and musculofascial implants disclosed herein can be innon-particulate form. When in non-particulate form, the implant can bein any desirable shape, e.g., a sheet, cube, sphere, or other desiredshape. In some embodiments, a non-particulate muscle or musculofascialimplant can have a thickness of up to about 20 mm for a single layer(e.g., about 2, 5, 10, 15, or 20 mm thick, or any thickness in between).The thickness of implants with multiple layers can depend upon thenumber of layers included.

Particulate implants (e.g., implants that have been cut, blended,cryofractured, or otherwise homogenized) can also be produced, and canbe stored dry (e.g., lyophilized) or suspended in a gel (e.g., gelatin),hydrogel, or aqueous solution (e.g., phosphate buffered saline or anyother biocompatible saline solution). Particulate implants can take theform of a powder or slurry that can be processed to have a putty-liketexture that is moldable into a variety of shapes. Particulate materialscan be used to produce slurry materials as discussed herein.

To improve the strength of the tissue composition, the tissuecomposition can comprise multiple layers of decellularized muscle. Inaccordance with the teachings herein, the tissue composition can containat least some of the myofibers normally found in an unprocessed musclesample. The tissue composition can be prepared by providing a group ofmuscle matrices and layering and joining the group of muscle matrices toproduce a multi-layer muscle matrix.

FIG. 2 is a side view of a layered muscle tissue composition 200according to various embodiments described herein. The layered muscletissue composition 200 can include multiple decellularized muscle matrixlayers 210 that are layered and joined. In some embodiments, the layersmay be joined by at least one spacer 220, but any suitable joiningmethod can be used, including, for example, mechanical anchors,biologically compatible adhesives, or cross-linking (e.g., chemical orenzymatic joining).

The multiple decellularized muscle matrix layers 210 can be formedsubstantially as described above. In some embodiments, the layeredmuscle tissue composition 200 can include muscle matrix layers 210 thathave different myofiber orientations. For example, one muscle matrixlayer 210 in the layered muscle tissue composition 200 can havecross-section myofiber orientation while a different muscle matrix layer210 can have longitudinal myofiber orientation. In some embodiments, thelayered muscle tissue composition 200 can include muscle matrix layers210 that have rotated myofiber orientations with respect to one another.For example, first and second muscle matrix layers 210 can havelongitudinal myofiber orientations but with the second muscle matrixlayer rotated with respect to the first muscle matrix layer such thatthe long axis of the myofibers in the first layer is perpendicular tothe long axis of the myofibers in the second layer.

If used, the spacers 220 can be evenly spaced or irregularly spacedthroughout the tissue composition. In some embodiments, the spacers 220can be rivets, screws, staples, tacks, or any other fashioning means. Insome embodiments, the spacers 220 can be biodegradable. When a layeredmuscle tissue composition 200 with spacers 220 such as rivets or tacksis used as part of a tissue implant during repair of abdominal wall andsimilar defects, the spacers 220 can provide relatively high initialload bearing capacity. Over time, the load bearing capacity can betransferred from the spacers 220 to the muscle portion of the implant(which is initially weaker) as muscle regeneration progresses and thespacer 220 degrades.

The muscle matrices can be layered and joined using a variety oftechniques. In some embodiments, the group of muscle matrices 210 can bejoined using dehydrothermal treatment or compression. In someembodiments, the group of muscle matrices 210 can be joined byinterlocking pieces of a first muscle matrix 210 with pieces of a secondmuscle matrix 210. In some embodiments, the spacers 220 can comprise anadhesive, cross-linking, or denaturation agent or can comprise a tissuematrix slurry. The tissue matrix slurry can include decellularizedmuscle tissue, decellularized musculofascial tissue, or otherdecellularized tissue such as decellularized dermal tissue.

In some embodiments, the spacers 220 can comprise transglutaminase toadhere adjacent tissue matrices to one another or to other components.Transglutaminases are enzymes expressed in bacteria, plants, and animalsthat catalyze the binding of gamma-carboxamide groups of glutamineresidues with amino groups of lysine residues or other primary aminogroups. In various embodiments, transglutaminases may be used tocatalyze binding of two or more muscle matrix layers to one another. Insome embodiments, transglutaminases can catalyze binding of collagen inone muscle matrix layer 210 to collagen in another muscle matrix layer210.

The layered muscle tissue composition 200 can have an increased strengthcompared to an individual muscle matrix layer 210. As described ingreater detail below with reference to FIGS. 9 and 10, a bilayer muscletissue composition can withstand a greater load than a single layer ofmuscle matrix.

Although FIG. 2 illustrates a layered muscle tissue composition 200including two decellularized muscle matrix layers 210, one of ordinaryskill in the art would appreciate that any number of muscle matrixlayers could be used to form the layered composition. For example, thecomposition can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50layers (or any number in between) depending on the desired use andneeded mechanical properties.

In some embodiments, the multi-layer muscle matrix can includeadditional muscle matrices, supporting layers, synthetic materials,metals, other biodegradable materials, or acellular tissue matrices. Incertain embodiments, the layered muscle tissue composition can includemuscle matrix layers attached to supporting layers using a particulateacellular tissue matrix.

FIG. 3A is a side view of a tissue composition 300 comprising musclematrix layers 310 and a slurry 320. The muscle matrix layer 310 may beprepared from a muscle matrix as described previously. In someembodiments, the slurry 320 can include a particulate acellular tissuematrix, including a particulate muscle matrix or other tissue matrixparticulate.

FIG. 3B is a side view of a tissue composition 300 comprising musclematrix layers 310, supporting layer 330, and a slurry 320. The musclematrix layer 310 may be prepared from a muscle matrix as describedpreviously. In some embodiments, the slurry 320 can include aparticulate acellular tissue matrix.

The supporting layers 330 may be any material capable of anchoring to orattached to (e.g., by being penetrated by or intermixed with pores ofthe layer 330) the slurry 320 including, but not limited to, metals,polypropylene, polytetrafluoroethylene, polyester, terephthalate,polyglycolide, or poly-4-hydroxybutyrate. In some embodiments, thesupporting layer 330 comprises a synthetic substrate. In furtherembodiments, the supporting layer 330 comprises a mesh. In someembodiments, the supporting layer 330 comprises a polypropylene mesh. Incertain embodiments, the supporting layer 330 can include at least oneof a porous foam, a planar mesh, a multifilament woven material, amonofilament woven material, multi-leveled layers, or multi-directionallayers. As shown, supporting layers is illustrated as spots, whichsignify cross-sectional portions of a mesh, e.g., a woven or knittedmesh, but other configurations of the supporting layer are within thescope of the supporting layer.

The supporting layer 330 can include pores or apertures 335 to improveconnectivity between muscle matrix layers 310 on opposite sides of thesupporting layer 330. In some embodiments, the pore size is at least 2mm. The use of pores or apertures 335 in the supporting layer 330 canencourage tissue ingrowth into the supporting layer 330. In someembodiments, the supporting layer 330 is embedded in the tissuecomposition 300.

In some embodiments, the slurry 320 can be used to join the musclematrix layers 310 to one another and/or to the supporting layer. In someembodiments, the slurry 320 can be used to join muscle matrices withsupporting layers 330. In some embodiments, the particulate acellulartissue matrix (ATM) in the slurry 320 may include dermal or muscletissue. In some embodiments, the particulate acellular tissue matrix maycomprise dried particles that are rehydrated for use in the slurry 320.In further embodiments, the particulate acellular tissue matrix has aporous structure. In some embodiments, the particulate acellular tissuematrix is cross-linked or otherwise stabilized. In some embodiments, thelayers 310, 320, and 330 of the tissue composition 300 are joined bycompression.

The slurry 320 may be disposed on a surface of the supporting layer 330to attach the supporting layer 330 to the muscle matrix layer 310. Anadditional slurry 320 may be disposed on an opposite surface of thesupporting layer 330 to attach a second muscle matrix layer 310.

As described above, the tissue composition can be substantially flat orcan be a flexible material that can be laid flat. Such compositions canbe used in a variety of situations to allow regeneration, augmentation,support, or other treatment of tissues. However, tissue compositions asdescribed herein may include other three-dimensional structures. In somecases, the tissue composition can include just a muscle matrix thatattaches to itself.

As shown in FIG. 4, a tissue composition 400 can be formed from a singlemuscle matrix layer 410, a supporting layer 430, and a slurry 420. Inaccordance with various embodiments, the supporting layer 430 caninclude one or more pores 435. The slurry 420 and supporting layer 430may be attached to the same side of the muscle matrix layer 410. Then,the muscle matrix layer 410 can be rolled such that the supporting layer430 is internal to the muscle matrix layer 410. When the muscle matrixlayer 410 and supporting layer 430 are rolled, a first surface of themuscle matrix layer 410 can attach to a second surface of the musclematrix layer through the pores 435. In this way, the supporting layer430 is not exposed to the exterior of the tissue composition 400 and themuscle matrix layer 410 can become bound to itself through the pores 435or apertures in the supporting layer 430.

In some embodiments, the slurry 320 (or 420) can includetransglutaminase (with or without particulate tissue matrix). Thetransglutaminases can catalyze binding of a supporting layer 330 to amuscle matrix layer 310 and/or another supporting layer 330. Forexample, the supporting layer 330 can be an organic or organic-derived(i.e., non-synthetic) material, and transglutaminase can cause formationof bonds between collagens of the supporting layer and muscle layer. Asanother example, a synthetic supporting layer 330 can be functionalizedto have exposed dipeptides on the surface.

In some embodiments, the slurry 320 can include transglutaminases andparticulate acellular tissue matrix. The transglutaminases can catalyzebinding of individual particles of the particulate acellular tissuematrix to one another and/or with the muscle layer(s). In someembodiments, transglutaminases can catalyze binding between collagens inindividual particles of the particulate acellular tissue matrix.

In accordance with the foregoing teachings, a variety oftransglutaminases can be used in the slurry 320 including any that arebiologically compatible, can be implanted in a patient, and havingsufficient activity to provide desired catalytic results within adesired time frame. Transglutaminases can include microbial, plant,animal, or recombinantly produced enzymes. Depending on the specificenzyme used, modifications such as addition of cofactors, control of pH,or control of temperature or other environmental conditions may beneeded to allow appropriate enzymatic activity. Microbialtransglutaminases can be effective because they may not require thepresence of metal ions, but any suitable transglutaminase may be used.

The use of transglutaminases to bind or join two or more materials canbe improved by causing partial denaturation of collagen at or near thesurface of the tissues, thereby making amine and acyl groups of collagenamino acids more accessible for enzymatic crosslinking. By partiallydenaturing collagen contained at or near the surface of a material, thedenatured collagen will remain connected to the fibrillar collagennetwork of the tissue product, and exogenous gelatin will not be neededto assist in binding with other materials such as other tissue productsor tissue at an implantation site. In accordance with variousembodiments, one or more of the muscle matrix layers, supporting layers,or slurry can be subjected to a denaturation process before applicationof transglutaminases thereto.

The denaturation process can be performed in a number of ways. Methodsfor controlled denaturation of the tissue matrix collagen may includephysical or mechanical processes (e.g., abrasion), thermal processes,chemical processes (e.g., acid, base or other protein denaturants),enzymatic denaturation, application of light (e.g., laser to heat orimpart energy), or combinations thereof.

Although the tissue composition 300 shown in FIG. 3B includes two musclematrix layers 310, one supporting layer 330, and two layers ofparticulate acellular tissue matrix slurry 320, one of ordinary skill inthe art would know that any number of each of these layers could be usedin combination to build up the tissue composition 300. In someembodiments, the tissue composition comprises at least one slurry 320adjacent to the at least one supporting layer opposite the at least onemuscle matrix layer. In some embodiments, the tissue composition caninclude at least one additional muscle matrix layer disposed adjacent tothe at least one additional dry particulate acellular tissue matrixopposite the at least one supporting layer.

Muscle and/or musculofascial implants, as described above, may bepackaged and/or stored as frozen, freeze-dried, hydrated, and/ordehydrated products. In certain embodiments, the packaged muscle ormusculofascial implants have reduced bioburden or are sterile. Incertain embodiments, a kit is provided, comprising one or more packagedmuscle implant(s) and instructions for preparing and/or using theimplant(s).

In some embodiments, a muscle or musculofascial implant can be treatedto reduce bioburden (i.e., the implant is aseptic or sterile). Suitablebioburden reduction methods are known to one of skill in the art, andmay include exposing the muscle or musculofascial implant to a compoundsuch as acids or to radiation or the use of ethylene oxide (EO) orsupercritical carbon dioxide treatments. Irradiation may reduce orsubstantially eliminate bioburden. In some embodiments, an absorbed doseof about 15-22 kGy of e-beam radiation is delivered in order to reduceor substantially eliminate bioburden. In various embodiments, a muscleimplant is exposed to between about 5 Gy and 50 kGy of radiation (e.g.,about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 kGy, or any value inbetween). Suitable forms of radiation can include gamma radiation,e-beam radiation, and X-ray radiation.

The multilayer implant(s) described herein can be implanted duringabdominal hernia repair. After implantation, the degree of myogenesisand fibroblast infiltration is measured and compared to myogenesis andfibroblast infiltration in the absence of an implant or in the presenceof an implant comprising intact muscle or fully decellularized tissue(e.g., decellularized tissue lacking any myofibers).

To evaluate the effectiveness of different implants and tissuecompositions, multiple types of implants were tested to measure loadcapacity. FIG. 5 is a bar graph illustrating the maximum load values ofporcine loin muscle cut in different directions and with differentorientations of myofibers. As described above, the myofibers in loinmuscle are primarily oriented obliquely with respect to the long axis ordirection of force generation of the muscle. Muscle cut along themyofibers demonstrated a maximum load of approximately 6 N/cm. Portionsof muscle cut along the longitudinal axis of the muscle or cutcross-sectionally (i.e., perpendicular to the longitudinal axis) werealso tested. It was discovered that cutting muscle along a specificorientation with respect to the myofibers increased the maximum loadthat could be applied to the resulting tissue. Specifically, across-sectional cut results in a maximum load of about 8 N/cm and alongitudinal cut results in a maximum load of about 10 N/cm. As shown bythese results, implant(s) prepared using muscle with cross-sectionallyoriented or longitudinally oriented myofibers may withstand a highermaximum load than muscle cut along (obliquely for loin) the myofibers.

FIG. 6 illustrates the results of testing the maximum load values ofporcine rectus abdominis muscle cut along or against the myofibers. Asdescribed previously, the myofibers in rectus abdominis muscle areprimarily oriented along the long axis or direction of force generationof the muscle. Muscle cut along the myofibers (i.e., along thelongitudinal direction) withstood a maximum load of about 6 N/cm. Musclecut against the myofibers (i.e., along a cross-sectional direction)withstood a maximum load of about 4 N/cm. Thus, in some embodiments,muscle cut along the myofibers may be able to withstand a higher loadthan muscle cut against the myofibers.

FIG. 7 illustrates the results of testing the maximum load values ofporcine loin muscle cut cross-sectionally and longitudinally and withand without fascia. Tissue cut along a cross-sectional directionwithstood a max load of about 7.5 N/cm without fascia and about 15 N/cmwith fascia. Tissue cut along a longitudinal direction withstood a maxload of about 10 N/cm without fascia and 25 N/cm with fascia. In variousembodiments, muscle with associated fascia attached can withstand ahigher load than muscle without fascia. Additionally, muscle cut along alongitudinally oriented direction can withstand a higher maximum loadthan muscle cut cross-sectionally. In some embodiments, muscle with theassociated fascia attached that was cut longitudinally can withstand thegreatest maximum load. In some embodiments, the greater the maximum loadthat can be withstood by the unprocessed muscle, the greater the maximumload that can be withstood by the processed muscle matrix.

FIG. 8 illustrates the results of testing the maximum load values ofexternal oblique muscles cut cross-sectionally or longitudinally andwith or without fascia. Tissue cut longitudinally withstood a max loadof about 17.5 N/cm without fascia and about 27.5 N/cm with fascia.Tissue cut cross-sectionally withstood a max load of about 12 N/cmwithout fascia and 21 N/cm with fascia. In accordance with variousembodiments, muscle with associated fascia attached may be able towithstand a higher load than muscle without fascia. Additionally, musclecut longitudinally can withstand a higher maximum load than muscle cutcross-sectionally. In accordance with various embodiments, muscle withassociated fascia attached that has been cut longitudinally canwithstand the greatest maximum load.

FIG. 9 illustrates the results of testing the maximum load values ofporcine muscle matrices comprising one layer and two layers derived fromcross-sectional cut loin muscle pieces. A single layer muscle matrixwithstood a maximum load of about 3.5 N/cm. Conversely, a bilayer musclematrix withstood a max load of about 8 N/cm. In some embodiments, musclesamples comprising more than one layer of muscle matrix can withstand agreater load than samples comprising only one layer of muscle matrix.

To determine if the strength of the muscle matrix derived from sectionscut along the longitudinal orientation is equivalent in all directions,samples were tested along two perpendicular directions. FIG. 10illustrates max load values of single and bilayer longitudinal cutporcine muscle matrix compositions measured in both X and Y directions.For single layer muscle matrices, one direction (e.g., the y-direction)yielded maximum load values that were about 3 times higher than in theother direction (e.g., the x-direction). These anisotropic results werepreserved in bilayer tissue compositions produced with two musclematrices that were aligned in the same direction. In contrast, bilayertissue compositions produced with two muscle matrices that were rotatedby 90 degrees with respect to one another showed similar maximum loadvalues when measured in either direction. In some embodiments,anisotropy can be maintained or eliminated in multi-layer tissuecompositions including muscle matrices by aligning or offsetting the cutdirection in individual muscle matrices with respect to one another.

Single layer implants derived from both longitudinal and cross-sectionalcut loin muscle can induce skeletal muscle repair in a gastrocnemiusdefect model. The data demonstrates that muscle regeneration does notdepend on the myofiber orientation of the implant.

In various embodiments, an implant comprising decellularized tissueharvested from the same region of connected muscle and fascia is used.In various embodiments, the fascia portion of the decellularized tissueprovides increases strength for the implant, as compared to an implantthat does not comprise decellularized fascia tissue. In someembodiments, the increased strength allows the implant to better resistthe tensile, torsional, and other forces the implant experiences duringthe regeneration process. In some embodiments, the decellularized fasciaportion of the implant provides a collagen scaffold into which nativecells (e.g., fibroblasts, etc.) can migrate, allowing for the remodelingof fascia along with the remodeled muscle induced by the decellularizedmuscle portion of the implant.

Disclosed herein are methods of making muscle and/or musculofascialimplants. In various embodiments, a muscle and/or musculofascial implantcomprises one or more decellularized muscle and/or fascial matrices thatare prepared by selecting suitable muscle and/or musculofascial samples,washing the samples to remove red blood cells and other debris, exposingthe muscle and/or musculofascial samples to trypsin, exposing the muscleand/or musculofascial samples to a decellularization solution,optionally contacting the decellularized muscle and/or musculofascialsamples with DNase and/or alpha-galactosidase, washing thedecellularized muscle and/or musculofascial samples, and, optionally,sterilizing the samples.

In various embodiments, the general steps involved in the production ofa decellularized muscle matrix include providing a sample of muscletissue, fascia tissue, or transition region tissue from a donor (e.g., ahuman cadaver or animal tissue source) and removing cellular materialunder conditions that preserve some or all of the biological and/orstructural functions of the extracellular matrix in the sample, as wellas at least some of the myofibers.

In some embodiments, a sample of muscle tissue and fascia tissue can beprovided and washed to remove any residual cryoprotectants, red bloodcells, and/or any other contaminants. Solutions used for washing can beany physiologically-compatible solution. Examples of suitable washsolutions include distilled water, phosphate buffered saline (PBS), orany other biocompatible saline solution.

In an embodiment, the at least one muscle sample and the least onefascia sample are contacted with a solution containing trypsin in orderto break down muscle fiber bundles (e.g., by cleaving myosin moleculesin the muscle fiber). In some embodiments, trypsin can facilitate thedecellularization process by increasing the rate and/or extent ofmyofiber breakdown and myocyte removal during subsequentdecellularization. In some embodiments, the muscle sample is exposed totrypsin at a concentration of about 10⁻¹⁰-0.5% (e.g., at about 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5 percent, or any percentage in between). In someembodiments, the trypsin concentration can range from 10⁻⁸-10⁻⁴%. Incertain embodiments, the muscle sample is exposed to trypsin for atleast about 15 minutes and/or up to a maximum of about 24 hours (e.g.,about 15, 30, 45, 60, 75, 90, 105, 120 minutes, 4 hours, 8 hours, 12hours, 24 hours or any time period in between). In certain embodiments,muscle samples including fascia can be exposed to trypsin for at leastabout 15 minutes and/or up to a maximum of about 48 hours (e.g., about15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes,105 minutes, 120 minutes, 4 hours, 8 hours, 12 hours, 24 hours, 48 hoursor any intermediate time).The length of time of trypsin exposure, and/orthe concentration of trypsin, can be adjusted in order to control theextent of myofiber removal from the muscle tissue so as to retain atleast some of the myofibers in the muscle sample and fascia sample aftertrypsinization and decellularization.

In various embodiments, the length of exposure and/or the concentrationof the decellularization solution and/or trypsin solution can beadjusted in order to control the extent of myofiber removal. In someembodiments, the duration and/or concentration are selected in order toremove about 20-80% of the myofibers normally found in the muscle sampleprior to trypsinization and decellularization. In certain embodiments,the duration and/or concentration are selected in order to remove about20, 30, 40, 50, 60, 70, or 80% of the myofibers (or any percentage inbetween). In some embodiments, about 20-80% of the myofibers are removedby exposing the muscle tissue sample to trypsin at a concentrationranging from 10⁻¹⁰-0.5% for 15 minutes to 48 hours and/or by exposingthe muscle tissue sample to about 0.1-2.0% of a decellularization agent(e.g., TRITON X-100™, sodium dodecyl sulfate, sodium deoxycholate,polyoxyethylene (20) sorbitan monolaurate, etc.) for 1-72 hours.

In various embodiments, about 20-80% of the myofibers normally found ina muscle sample are removed by controlling the tissue to volume ratio(e.g., the mass of tissue per volume of solution containing trypsinand/or decellularizing agents). In some embodiments, a lowertissue/volume ratio increases the efficiency of the myofiber removalprocess, thus resulting in a muscle matrix that retains fewer intactmyofibers. In other embodiments, a higher tissue/volume ratio reducesthe efficiency of the myofiber removal process, thus resulting in amuscle matrix that retains more intact myofibers.

In some embodiments, after decellularization, the muscle and/ormusculofascial tissue is washed thoroughly. Any physiologicallycompatible solutions can be used for washing. Examples of suitable washsolutions include distilled water, phosphate buffered saline (PBS), orany other biocompatible saline solution. In some embodiments, the washsolution can contain a disinfectant. In certain, embodiments, thedisinfectant is peracetic acid (PAA), for example at a concentration of0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, or 0.5% (or any percentage inbetween). In certain embodiments, e.g., when xenogenic or allogenicmaterial is used, the decellularized muscle tissue is treated (e.g.,overnight at room temperature) with a deoxyribonuclease (DNase)solution. In some embodiments, the tissue sample is treated with a DNasesolution prepared in a DNase buffer. Optionally, an antibiotic solution(e.g., Gentamicin) may be added to the DNase solution. Any suitableDNase buffer and/or antibiotics can be used, as long as the bufferand/or antibiotic provides for suitable DNase activity.

The preceding examples are intended to illustrate and in no way limitthe present disclosure. Other embodiments of the disclosed devices andmethods will be apparent to those skilled in the art from considerationof the specification and practice of the devices and methods disclosedherein.

The above description and embodiments are exemplary only and should notbe construed as limiting the intent and scope of the invention.

What is claimed is:
 1. A tissue composition comprising: multiple layersof decellularized muscle matrix that contain at least some of themyofibers normally found in an unprocessed muscle sample, and one ormore binding layers comprising particulate acellular tissue matrix (ATM)to attach the multiple layers of decellularized muscle matrix to oneanother.
 2. The tissue composition of claim 1, wherein the particulateATM has been treated with a transglutaminase.
 3. The tissue compositionof claim 1, wherein a direction in which the first decellularized musclematrix layer was cut from a muscle is aligned with a direction in whichthe second decellularized muscle matrix layer was cut from a muscle. 4.The tissue composition of claim 1, wherein a direction in which thefirst decellularized muscle matrix layer was cut from a muscle is offsetfrom a direction in which the second decellularized muscle matrix layerwas cut from a muscle.
 5. The tissue composition of claim 1, wherein theparticulate ATM comprises dermal or muscle tissue matrix.
 6. The tissuecomposition of claim 1, wherein the particulate ATM comprises rehydratedparticles.
 7. The tissue composition of claim 1, wherein the particulateATM comprises a porous structure.
 8. The tissue composition of claim 1,wherein the particulate ATM in the binding layer is cross-linked.
 9. Thetissue composition of claim 1, wherein the particulate ATM comprisesmuscular tissue matrix.
 10. The tissue composition of claim 1, whereinthe layers of decellularized muscle matrix are further joined byinterlocking pieces of the layers of decellularized muscle matrix. 11.The tissue composition of claim 1, wherein the layers of decellularizedmuscle matrix comprise porcine muscle tissue.
 12. A tissue composition,comprising: a muscle matrix layer; and a binding layer comprisingparticulate acellular tissue matrix (ATM) applied to the muscle matrixlayer, wherein the muscle matrix layer has been rolled such that atleast a portion of a first surface of the muscle matrix layer attachesto a portion of a second surface of the muscle matrix layer.
 13. Thetissue composition of claim 12 further comprising a supporting layer incontact with the first surface of the muscle matrix layer.
 14. Thetissue composition of claim 13, wherein at least the portion of thefirst surface attaches to the portion of the second surface through oneor more pores in the supporting layer.
 15. The tissue composition ofclaim 13, wherein the supporting layer comprises a synthetic substrate.16. The tissue composition of claim 13, wherein the supporting layercomprises a polypropylene or metal mesh.
 17. The tissue composition ofclaim 12, wherein the particulate ATM has been treated with atransglutaminase.
 18. The tissue composition of claim 12, wherein theparticulate ATM comprises dermal or muscle tissue matrix.
 19. The tissuecomposition of claim 12, wherein the particulate ATM comprisesrehydrated particles.
 20. The tissue composition of claim 12, whereinthe particulate ATM comprises a porous structure.
 21. The tissuecomposition of claim 12, wherein the particulate ATM in the bindinglayer is cross-linked.